We propose experiments to test the hypothesis that oligonucleotide-mediated triple helix formation can stimulate homologous recombination in mammalian cells. The broad, long- term objective of these studies is to learn how triplex-forming oligonucleotides (TFOs) can be used as a tool for the genetic manipulation of mammalian cells for the ultimate purpose of gene therapy. Initial work has shown that TFOs can stimulate recombination between duplicated genes in an SV40 vector in mammalian cells, and preliminary studies with repair-deficient cell lines indicate that this induction may be dependent on nucleotide excision repair (NER). In recent studies using a mouse LTK- cell line carrying two mutant copies of the thymidine kinase (TK) gene as direct repeats in a single locus, we were able to demonstrate triplex-induced recombination at a chromosomal site. In the proposed work, factors that influence TFO-induced recombination will be studied using the established LTK- cell assay, with the focus on intracellular TFO delivery and TFO chemical modification. Novel recombination substrates will be developed to report recombination induced either by TFOs of by double-strand breaks generated by the I-Sce I nuclease. These novel vectors will be used in repair-deficient cell lines, with an emphasis on determining the influence of NER, transcription- coupled repair, and the Rad51-associated pathway of homologous recombination on triplex-induced recombination. Based on preliminary work demonstrating triplex-induced recombination in human cell-free extracts, we will further probe the roles of specific repair and recombination proteins, using a battery of antibodies and purified proteins to manipulate the in vitro reaction. These experiments will serve as a basis for designing additional cell lines and experimental protocols to test triplex-induced recombination between a chromosomal site and a separate homologous donor DNA fragment, in situations where the TFO is directed to bind either within or outside of the target gene. The possibility of a further enhancement in gene targeting will be tested using a novel strategy in which the TFO is covalently tethered to a short donor DNA fragment. With this hybrid molecule, target site recognition can be mediated by triplex formation, thereby positioning the donor fragment for efficient recombination and information transfer. We will build on positive initial results to pursue a systematic examination of this approach, using both episomal and chromosomal reporter gene constructs in mammalian cells. This work will provide the foundation for future efforts to correct mutations in disease- related human genes.